Deformation and flow of red blood cells in a synthetic lattice: evidence for an active cytoskeleton.

We introduce the use of microfabrication techniques to construct on a silicon wafer a synthetic capillary bed with 2.5- to 4-micron (mu)-wide channels. Establishment of a fluid pressure gradient allowed us to observe simultaneously using optical microscopy hundreds of cells flowing through the bed at physiological speeds. We find a large distribution of mobilities among red cells flowing through the structure; smaller channels provide a greater impedance to flow than larger ones, indicating that kinetic drag variations provide the origin of the distribution. The mobility of a particular cell is not correlated with the cell diameter but appears to be inversely correlated with intracellular calcium concentration of the cell, as determined by fluorescence of the calcium-binding dye fluo-3 AM. Also, we are able to use the parallel processing nature of our arrays to observe isolated events where the rigidity of the red cell seems to change suddenly over several orders of magnitude as it blocks a channel in the array.     

Comparative evolutionary rates of introns and exons in murine rodents

Analysis of DNA sequences of 132 introns and 140 exons from 42 pairs of orthologous genes of mouse and rat was used to compare patterns of evolutionary change between introns and exons. The mean of the absolute difference in length (measured in base pairs) between the two species was nearly five times as high in the case of introns as in the case of exons. The average rate of nucleotide substitution in introns was very similar to the rate of synonymous substitution in exons, and both were about three times the rate of substitution at nonsynonymous sites in exons. G+C content of introns and exons of the same gene were correlated; but mean G+C content at the third positions of exons was significantly higher than that of introns or positions 1–2 of exons from the same gene. G+C content was conserved over evolutionary time, as indicated by strong correlations between mouse and rat; but the change in G+C content was greatest at position 3 of exons, intermediate in introns, and lowest at positions 1–2 in introns. Received: 23 December 1996 / Accepted: 1 April 1997     

Characterization of cyclic AMP-binding proteins in rat Sertoli cells using a photoaffinity ligand

Summary Protein-bound cyclic AMP (cAMP) levels in cultured rat Sertoli cells have been determined after exposure to follicle-stimulating hormone (FSH) and agents which elevate intracellular cAMP or mimic cAMP action. Changes in the content of protein-bound cAMP were correlated with changes in receptor availability determined by measuring [³H] cAMP binding. Using the photoaffinity analog of cAMP, 8-N₃ [³²P] cAMP, two major cAMP-binding proteins in Sertoli cell cytosol, with molecular weights of 47 000 and 53 000 daltons, were identified as regulatory subunits of type I and type II cAMP-dependent protein kinases, respectively. Densitometric analysis of autoradiograms demonstrated differential activation of the two isozymes in response to treatment with FSH and other agents. Results of this study demonstrate the value of measuring changes in protein-bound cAMP and the utility of the photoaffinity labeling technique in correlating hormone-dependent processes in which activation of cAMP-dependent protein kinase occurs.     

Hyperthermia,antipyretics and function of polymorphonuclear leukocytes.

Whether hyperthermia (temperature, 40 degrees C), salicylates, acetaminophen or phenacetin has an adverse effect on polymorphonuclear leukocyte (PMNL) function was examined. Migration experiemnts were carried out in Boyden chambers with bacterial chemotactic factor as the attract, and bactericidal assays were done with Staphylococcus aureus and serum from an AB blood group donor as a source of opsonins. PMNL viability was determined by the trypan blue exclusion method. Neither hyperthermia nor any of the drugs tested affected PMNL viability adversely, but sodium salicylate and phenacetin suppressed PMNL migration. Early staphylococcal killing was greater at 40 degrees C; however, after 2 hours the converse was true. Bactericidal activity was suppressed by acetylsalicylic acid, sodium salicylate and phenacetin. Hence it appears PMNL function is similar at 37 degrees and 40 degrees C but that some commonly used antipyretics have an adverse effect on PMNL activity.     

The pathways of assimilation of 13NH4+ by the cyanobacterium, Anabaena cylindrica.

The principal initial product of metabolism of 13N-labeled ammonium by Anabaena cylindrica grown with either NH4+ or N2 as nitrogen source is amide-labeled glutamine. The specific activity of glutamine synthetase is approximately half as great in NH4+-grown as in N2-grown filaments. After 1.5 min of exposure to 13NH4+, the ratio of 13N in glutamate to 13N in glutamine reaches a value of approximately 0.1 for N2- and 0.15 for NH4+-grown filaments, whereas after the same period of exposure to [13N]N2, that ratio has reached a value close to unity and is rising rapidly. During pulse-chase experiments, 13N is transferred from the amide group to glutamine into glutamate, and then apparently into the alpha-amino group of glutamine. Methionine sulfoximine, an inhibitor of glutamine synthetase, inhibits the formation of glutamine. In the presence of the inhibitor, direct formation of glutamate takes place, but accounts for only a few per cent of the normal rate of formation of that amino acid; and alanine is formed about as rapidly as glutamate. Azaserine reduces formation of [13N]glutamate approximately 100-fold, with relatively little effect on the formation of [13N]glutamine. Aminooxyacetate, an inhibitor of transaminase reactions blocks transfer of 13N to aspartate, citrulline, and arginine. We conclude, on the basis of these results and others in the literature, that the glutamine synthetase/glutamate synthase pathway mediates most of the initial metabolism of ammonium in A. cylindrica, and that glutamic acid dehydrogenase and alanine dehydrogenase have only a very minor role.     

IN VIVO RNA SYNTHESIS WITHIN THE RAT NODOSE GANGLIA

Abstract— The in vivo synthesis of RNA in the rat nodose ganglia has been studied following the intravenous introduction of ortho-[³²P]phosphate. Analysis of the labelled RNA upon 2.2% polyacrylamide gels was performed. Rapidly-labelled heterodisperse RNA. with a size range of 10S-30S was detected after 3 h exposure to the isotope. Two peaks, of size 28S and 12S-14S. were also observed. The former is thought to be processed 28S rRNA whereas the latter is consistent with its designation as 'message-like' RNA (mlRNA). A rapidly turning-over phosphorylated non-nucleic acid contaminant prevented clear interpretation of the 4S region of the gel at short labelling times. However, this material was not present when the exposure time was increased to 24 h. At this time, only the stable RNA species 28S and 18S rRNA and 4S tRNA, were detected.     

Properties of carnitine transport in rat kidney cortex slices

The properties of carnitine transport were studied in rat kidney cortex slices. Tissue: medium concentration gradients of 7.9 for L-[methyl-¹⁴C]carnitine were attained after 60-min incubation at 37°C in 40 μM substrate. L- and D-carnitine uptake showed saturability. The concentration curves appeared to consist of (1) a high-affinity component, and (2) a lower affinity site. When corrected for the latter components, the estimated K_m for L-carnitine was 90 μM and $\text{V = 22}\text{nmol/min}$ per ml intracellular fluid; for D-carnitine, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 166}\text{μM}$ and $\text{V = 15}\text{nmol/min}$ per ml intracellular fluid. The system was stereospecific for L-carnitine. The uptake of L-carnitine was inhibited by (1) D-carnitine, γ-butyrobetaine, and (2) acetyl-L-carnitine. γ-Butyrobetaine and acetyl-L-carnitine were competitive inhibitors of L-carnitine uptake. Carnitine transport was not significantly reduced by choline, betaine, lysine or γ-aminobutyric acid. Carnitine uptake was inhibited by 2,4-dinitrophenol, carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$, N₂ atmosphere, KCN, $\text{N-}\text{ethylmaleimide}$, low temperature (4°C) and ouabain. Complete replacement of Na⁺ in the medium by Li⁺ reduced L- and D-carnitine uptake by 75 and 60%, respectively. Complete replacement of K⁺ or Ca²⁺ in the medium also significantly reduces carnitine uptake. Two roles for the carnitine transport system in kidney are proposed: (1) a renal tubule reabsorption system for the steady-state maintenance of plasma carnitine; and (2) maintenance of normal carnitine levels in kidney cells, which is required for fatty acid oxidation.     

THE DIRECT MEASUREMENT OF THE AXOPLASMIC TRANSPORT OF INDIVIDUAL RNA SPECIES: TRANSFER BUT NOT RIBOSOMAL RNA IS TRANSPORTED

Analysis of chick retinal and tectal RNA revealed that in addition to the major cytoplasmic RNAs (rRNA and tRNA), a number of the small mol wt nuclear RNAs (snRNAs) can also be detected. Subfractionation data indicated that one of these molecules, DD′, is of at least 95% nuclear location within the retina. Thus, very little, if any, of the retinal DD′ is available for axoplasmic transport from the retina into the optic nerve and tectum. Following intraocular injection of [³H]uridine, considerable incorporation of isotope into DD′ was observed within the optic tectum after 4, 8 and 16 days. This result indicates the presence of considerable local (i.e. tectal) synthesis. The specific activities of 29S, 18S and 5S rRNA and 4s tRNA relative to that of DD′ were measured in the optic tectum 8 and 16 days after the intraocular introduction of [³H]uridine. The same measurements were also made in intracranially injected animals. While the 29S/DD′, 18S/DD′ and 5S/DD′ specific activity ratios obtained were independent of the injection route, the 4S/DD′ ratio obtained from intraocularly injected animals was significantly greater (at least 2-fold) than that obtained from intracranially injected animals. Similar analysis was also performed with the optic nerve complex at 16 days post-injection with identical results. These results demonstrate that tRNA, but not rRNA, is transported from the retina into the optic nerve and tectum in the 2-day-old chicken.     

A paradigm for axonal transport

Axonal transport has been extensively studied for a period of 20–30 years, but there is still no general consensus concerning the mechanism by which this transport process operates. An important development in this regard is the recent studies in the physical biochemistry group in the Department of Biochemistry at Monash University where it has been demonstrated that ordered flows may be generated spontaneously in polymer systems under non-equilibeium conditions. The new phenomenon exhibits many novel features, particularly with respect to polymer transport, which bear marked similarity to the behaviour of components in axonal transport. This article sets out to essentiallybring to the attention of those in the neurosciences some of the properties of ordered structured flows in polymer solutions. These properties may generate a different view in the understanding of the mechanism of axonal transport.     

Increased in vitro labeling of stable RNA within the rat nodose ganglion following abdominal vagotomy

An in vitro procedure for labeling of RNA in the excised rat nodose ganglion was used to evaluate the changes in incorporation of [³H]uridine into ganglionic RNA following transection of the abdominal vagus nerves. Significant increases in the incorporation into 28S, 18S and 4S RNA were observed at 1 day after injury, which were maximal at 4 days before returning to unoperated control level by 7 days. A second transient increase in the labelling of these RNA species occurred between 9 and 11 days after injury. Comparison of the time course of these increases with those seen previously following cervical vagus nerve crush injury indicate that the time of onset of the increase in incorporation is independent of the site of injury, but that the maximal response is delayed by 1 day with the more distal lesion. These data are consistent with the existence of separate signals for initiating and modulating the cell body response to axon injury, which are transported retrogradely from the site of injury at rates exceeding the slow component of axoplasmic transport.